Bilirubin is the principal pigment in blood serum and bile. It is derived from the porphyrin fraction of hemoglobin which is released by the disintegration of erythrocytes. Any disturbance that causes increased destruction of red blood cells results in an increase in bilirubin.
The determination of serum bilirubin is a necessary part of the routine evaluation of liver disease and the appearance of bilirubin in the urine can be the earliest sign of hepatobiliary disease.
A number of methods of assaying or quantitatively measuring bilirubin in the serum are based on the coupling of bilirubin with diazotized sulfanilic acid to form a red pigment in neutral solutions and a blue pigment in strongly acid or alkaline solutions. Van den Bergh and Snapper described the use of this color reaction for quantitative measurements of serum bilirubin in their article in Dent. Arch. Klin. Med. 110, 540-561 (1913). Later, Jendrassik and Grof combined caffeine-sodium benzoate with sodium acetate, diazotized sulfanilic acid, and formed the azobilirubin color within 10 minutes. Biochem. Z. 297, 81-89 (1938).
At the present time, the most commonly used method for measuring direct bilirubin is that proposed by Michaelsson which is based on the Jendrassik-Grof principle. Scand. J. Clin. Lab. Invest. 13, Suppl. 56, 1; (1960). In the Michaelsson method hydrochloric acid (HCl) is used to prevent unconjugated bilirubin from reacting; ascorbic acid is added to destroy the excess diazo reagent; and the caffeine reagent is added at the end to prevent overestimation of the direct bilirubin. The molar absorptivity of the alkaline azopigment is considerably higher in the absence of caffeine (about 12%) than in its presence, and since caffeine is needed for the reaction of the standard, it must also be added in the direct reaction.
In the Michaelsson method, even at a concentration as low as 0.6 g/L, hemoglobin causes a 21% suppression of the direct bilirubin value. According to Michaelsson, the average hemoglobin concentration in samples obtained by a heel stick is about 1.2 g/L and it is not unusual to see hemoglobin levels of 3 g/L. The fact that the ascorbic acid fails to counteract the effect of hemoglobin indicates that bilirubin or azobilirubin is being destroyed before ascorbic acid is added.
The mechanism of hemoglobin interference appears to be as follows: At an acid pH, hemoglobin is oxidized to hematin and, H.sub.2 O.sub.2 or some form of peroxide is released during the reaction. The peroxide in the presence of heme protein, which acts as a pseudo-peroxidase, oxidizes conjugated bilirubin to a diazo-negative compound. Any unoxidized bilirubin reacts upon addition of the diazo reagent to give the azopigment and no further destruction of the azopigment occurs. It is highly probable that the oxidation product of conjugated bilirubin is conjugated biliverdin.